1. Field of the Invention
The present invention relates to a novel process for the preparation of combretastatins and of their derivatives.
2. Description of the Art
The term xe2x80x9ccombretastatinsxe2x80x9d or xe2x80x9cstilbene derivativesxe2x80x9d is understood to mean the derivatives of following general formula (I): 
in which A represents a hydroxyl group or an amino group, and their pharmaceutically acceptable salts.
Mention may be made, among the salts, of the hydrochloride, acetate, phosphate or methanesulphonate. When A in the compound is an amino group, it can also be coupled to amino acids to result in amides, and their pharmaceutically acceptable salts.
The synthesis of stilbene derivatives or combretastatins, which can be in the form of a pharmaceutically acceptable salt, and the pharmaceutical compositions which comprise them are disclosed in U.S. Pat. Nos. 4,996,237, 5,525,632, 5,731,353 and 5,674,906. These patents disclose combretastatins and their metabolites and disclose their in vitro oncologic activity.
According to these patents, combretastatins are prepared from (3,4,5-trimethoxybenzyl)triphenylphosphonium salts, which are condensed with a 3-nitro- or 3-hydroxy-4-methoxybenzaldehyde (the hydroxyl group of which is protected) in the presence of sodium hydride or of lithium derivatives, and then the derivative obtained, when it is nitrated, is reduced in the presence of zinc.
The isomer with the cis configuration is subsequently prepared by the action of light or by chromatographic separation of the mixture.
The present invention provides novel processes for the preparation of combretastatins or stilbene compounds of formulae (I) and (III) 
wherein A is 
and
PG is a protecting group selected from the group consisting of tert-butoxycarbonyl, benzyloxycarbonyl and 9-fluorenylmethyloxycarbonyl, or the pharmaceutically acceptable salts thereof. In addition, the novel intermediate compound of formula III is disclosed and claimed.
A first process route V0 1 for the preparation of derivatives of formula (I) for which A represents an amino group has first been discovered, which process is an improvement to the process disclosed in the abovementioned patents, which consists, after the Wittig condensation of (3,4,5-trimethoxybenzyl)triphenylphosphonium bromide or chloride and 3-nitro-4-methoxybenzaldehyde, in carrying out reduction of the nitro group with of iron, instead of the zinc as is used in the prior publications, which makes it possible to achieve an overall reaction yield, with respect to the aldehyde charged, of 60% (the yield with respect to the aldehyde charged in U.S. Pat. No. 5,525,632 is between 21% and 33%).
The first process route V0 2 consists in condensing 3,4,5-trimethoxybenzaldehyde with (4-methoxy-3-nitrobenzyl)triphenylphosphonium bromide or chloride. For both these first two processes routes V0 1 and V0 2, the reaction is carried out in the presence of a base chosen in particular from potassium tert-butoxide, sodium tert-pentoxide, sodium hydride, butyllithium, LDA (lithium dilsopropylamide), sodium methoxide, potassium carbonate or alkaline derivatives of hexamethyldisilazane.
This reaction is carried out in various solvents, such as ethers (THF), polar aprotic solvents (acetonitrile, NMP, DMF, DMSO, and the like), alcohols, aromatic solvents or water, at a temperature which will be adjusted by a person skilled in the art to the base used and to the solvent used.
This reaction, as regards the first process route V0 2, is described in particular in the publication by K. G. Piney which appeared in Bioorg. Med. Chem., 8(2000), 2417-2425.
2-Methoxy-5-[2-(3,4,5-trimethoxyphenyl)vinyl]nitrobenzene is reduced according to the improved process of the invention by the action of iron. It is advantageous to use an amount of iron in excess if complete conversion of the starting material is desired. This excess is advantageously greater than 2 equivalents per one mol of starting nitro derivative.
It has been shown that the same stage, carried out in the presence of zinc in acetic acid, a conventional solvent for reductions with zinc, does not make it possible to obtain complete reaction (in U.S. Pat. No. 5,525,632, the yield of the reduction carried out on the pure Z isomer varies between 46% and 66%) and, moreover, that the amounts of zinc used are large and consequently result in considerable industrial waste. Furthermore, the process generates a large amount of xe2x80x9cazoxe2x80x9d compound resulting from coupling between the amino formed and the nitroso intermediate in the reduction.
Reduction with nascent hydrogen, generated by ammonium formate in the presence of a conventional catalyst, such as palladium or platinum, leads to high isomerization of the double bond to the undesirable E isomer and to partial saturation of the double bond.
The abovementioned Piney publication describes the reduction by sodium hydrosulphite of a pure nitro Z isomer, obtained after chromatography and recrystallization, leading to an amino Z isomer with a yield of only 37%.
Hydrogenations with molecular hydrogen, catalysed by platinum or palladium, are rarely complete and result in particular in the saturation of the ethylenic double bond.
A second process has also been found which avoids the intermediate reduction stage necessary when starting from a nitro derivative. This is because it is much more economical to condense, according to a first method of carrying out this second process, a (3,4,5-trimethoxybenzyl)triphenylphosphonium bromide or chloride with 3-amino-4-methoxybenzaldehyde or, according to a second method of carrying out this second process, condensing 3,4,5-trimethoxybenzaldehyde with a (3-amino-4-methoxybenzyl)triphenyl-phosphonium salt.
This second process according to its two alternative forms requires a stage in which less in the way of CMR (Cancerogenic, Mutagenic or Reproductive) toxic products are given off in comparison with the first processes routes V0 1 and V0 2, which is a considerable advantage at the industrial level from the viewpoint of safety and production cost.
According to the second process route V0 3 for implementing the invention, the (3,4,5-trimethoxybenzyl)triphenylphosphonium salt and 3-amino-4-methoxybenzaldehyde are brought together and the reaction is carried out, preferably, in the presence of a base chosen in particular from potassium tert-butoxide, sodium tert-pentoxide, sodium hydride, butyllithium, LDA, sodium methoxide, potassium carbonate or alkaline derivatives of hexamethyldisilazane. Use is preferably made of sodium methoxide.
This reaction is carried out in various solvents, such as ethers (THF), polar aprotic solvents (acetonitrile, NMP, DMF, DMSO, and the like), alcohols, aromatic solvents or water, at a temperature which will be adjusted by a person skilled in the art to the base used and to the solvent used.
The reaction temperature will be adjusted by a person skilled in the art as to the base used. When methoxide is used, the reaction temperature is preferably between 0xc2x0 C. and 10xc2x0 C. After reaction, the base used is neutralized with an acid in aqueous solution, the organic phase is washed and concentrated, and the expected product is obtained after chromatographing the crude concentrate.
According to the second process route V0 4 for implementing the invention, in which the (3-amino-4-methoxybenzyl)triphenylphosphonium salt and 3,4,5-trimethoxybenzaldehyde are brought together, the reaction is preferably carried out in the presence of a base chosen in particular from potassium tert-butoxide, sodium tert-pentoxide, sodium hydride, butyllithium, LDA, sodium methoxide, potassium carbonate or alkaline derivatives of hexamethyldisilazane. Use is preferably made of sodium methoxide.
This reaction is carried out in various solvents, such as ethers (THF), polar aprotic solvents (acetonitrile, NMP, DMF, DMSO, and the like), alcohols, aromatic solvents or water, at a temperature which will be adjusted by a person skilled in the art to the base used and to the solvent used.
The reaction temperature will be adjusted by a person skilled in the art to the base used. When methoxide is used, the reaction temperature is preferably between 0xc2x0 C. and 10xc2x0 C. After reaction, the base used is neutralized with an acid in aqueous solution, the organic phase is washed and concentrated, and the expected product is obtained after chromatographing the crude concentrate.
The derivative obtained according to the second process route V0 3 or V0 4 or during the second stage of the first process route V0 1 or V0 2 has the formula (IIa): 
It is advantageous, when it is desired to couple serine with the compound of formula (IIa), to use L-serine doubly protected on the nitrogen of the serine and on the hydroxyl functional group of general formula (IIb) 
where PG represents a protective group for the amine functional group well known to a person skilled in the art, to give a novel intermediate of following general formula (III): 
which is subsequently cleaved, preferably simultaneously with the opening of the ring, by acid hydrolysis according to a deprotection reaction well known to a person skilled in the art. Preferably, the PG group of the formulae (IIb) or (III) represents a protective group selected from the following groups: tert-butoxycarbonyl, benzyloxycarbonyl (CBZ) or 9-fluorenylmethyloxycarbonyl (FMOC).
The compound of formula (III) above is novel and is claimed as such.
The condensation is advantageously carried out in the presence of EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) or in the presence of DCC (dicyclohexylcarbodiimide) and of HOBT (hydroxybenzotriazole) or in the presence of DCC (dicyclohexylcarbodiimide) and of HOSU (N-hydroxysuccinimide) or, finally, in the presence of TOTU (O-[(ethoxycarbonyl)cyanomethyleneamino]-N,N,Nxe2x80x2,Nxe2x80x2-tetramethyluronium tetrafluoroborate) or of HBTU (O-benzotriazol-1-yl-N,N,Nxe2x80x2,Nxe2x80x2-tetramethyluronium hexafluorophosphate) or of N,N-carbonyldiimidazole. The reaction is preferably carried out in a solvent which is inert with respect to the reaction, which solvent is chosen in particular from polar aprotic solvents, such as acetonitrile, dimethylformamide, tetrahydrofuran or chlorinated aliphatic solvents, such as dichloromethane, or, finally, esters.
The coupling to the derivative of formula (IIa) can also be carried out by the action of a mixed anhydride, synthesized in situ between a chloroformate or a carboxylic acid chloride, for example pivaloyl chloride, and doubly protected L-serine of formula (IIb), in the presence of a tertiary base of the NMM (N-methylmorpholine) type in various solvents which are inert with respect to the reaction such as, for exampe, esters, ethers, chlorinated solvents, acetonitrile, and the like. The mixed anhydride is preferably prepared at a temperature of between 0xc2x0 C. and 10xc2x0 C. and then the reaction is carried out at ambient temperature. After reaction, the reaction mixture is hydrolysed with an aqueous solution, then the phases are separated and the organic phase is washed with aqueous base.
The double deprotection of the compound of formula (III) is carried out by the action of an organic or inorganic acid. Use is preferably made of concentrated aqueous hydrochloric acid in an alcoholic medium. The reaction temperature is, according to a better means of implementation of the invention, preferably between 50xc2x0 C. and 70xc2x0 C.
The invention will be more fully described with the help of the following examples, which must not be regarded as limiting the invention.
The composition of the mixtures, the monitoring and the progression of the reactions, and the yield of the unisolated products/intermediates and their assays are determined by HPLC (High Performance Liquid Chromatography) analysis. HPLC conditions: columnxe2x80x94octadecyl silicagel; detectionxe2x80x94UV 242 nm; mobile phasexe2x80x94water, trifluoroacetic acid, acetonitrile. Thin layer chromatographic analysis (TLC) was performed using silica gel plates with cyclohexane/ethyl acetate.